This application is a 371 of PCT/EP00/02121 filed on Mar. 10, 2000.
The present invention relates to a method for determining the enantioselectivity of kinetic resolution processes and of asymmetrically proceeding reactions of prochiral compounds bearing enantiotopic groups by using isotope-labeled substrates so that the reaction products can be quantitatively determined with an isotope-specific detection system, e.g., an ESI mass spectrometer. In connection with an automated sampler the method can be employed for high throughput screening. The relevant enantioselective conversions can be induced by chiral homogeneous or heterogeneous catalysts, biocatalysts or stoichiometric amounts of optically active agents.
The development of effective methods for generating extensive libraries of chiral chemical catalysts by methods of combinatorial chemistry [a) G. Liu, J. A. Ellman, J. Org. Chem. 1995, 60, 7712-7713; b) K. Burgess, H.-J. Lim, A. M. Porte, G. A. Sulikowski, Angew. Chem. 1996, 108, 192-194; Angew. Chem., Int. Ed. Engl. 1996, 35, 220-222; c) B. M. Cole, K. D. Shimizu, C. A. Krueger, J. P. A. Harrity, M. L. Snapper, A. H. Hoveyda, Angew. Chem. 1996, 108, 1776-1779; Angew. Chem., Int. Ed. Engl. 1996, 35, 1668-1671; d) C. Gennari, H. P. Nestler, U. Piarulli, B. Salom, Liebigs Ann./Recl. 1997, 637-647] or for the preparation of libraries of enantioselective biocatalysts by in-vitro evolution [M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963; Angew. Chem., Int. Ed. Engl. 1997, 36, 2830-2932] is currently under investigation. A critical aspect of the success of these novel technologies is the existence of effective and rapid methods for the screening of the enantioselective catalysts or biocatalysts from the respective catalyst libraries. While many effective methods for the screening of large libraries of biologically active compounds are available in the combinatorial chemistry of active substances [a) F. Balkenhohl, C. von dem Bussche-Hxc3xcnnefeld, A. Lansky, C. Zechel, Angew. Chem. 1996, 108, 2436-2488; Angew. Chem., Int. Ed. Engl. 1996, 35, 2288-2337; b) J. S. Frxc3xcchtel, G. Jung, Angew. Chem. 1996, 108, 19-46; Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42; c) Chem. Rev. 1997, 97 (2), 347-510 (special edition about combinatorial chemistry); d) S. R. Wilson, A. W. Czarnick, Combinatorial Chemistry: Synthesis and Application, Wiley, N.Y., 1997], the development of methods for the high throughput screening of enantioselective catalysts, biocatalysts or optically active agents is still at the beginning. The determination of the enantiomeric excess (ee) of the products of stereo-selective conversions is normally effected classically by means of gas or liquid chromatography on chiral stationary phases [G. Schomburg, Gaschromatographie: Grundlagen, Praxis, Kapillartechnik, 2nd Ed., VCH, Weinheim, 1987; K. K. Unger, Packings and stationary phases in chromatographic techniques, Series Chromatographic science; Vol. 47, Marcel Dekker, New York, 1990]. Although precise ee values can be determined thereby, such conventional methods have a disadvantage in that only a limited number of samples can be examined per unit time since the times required for analysis depend on the respective retention times.
The first suggestions for solving analytical problems of this kind have been made recently. Thus, for example, in the scope of a study on the in-vitro evolution of enantioselective lipases, a relatively rough test method has been developed according to which the course of enantioselective hydrolyses of chiral carboxylate esters can be determined [M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963; Angew. Chem., Int. Ed. Engl. 1997, 36, 2830-2932]. Thus, the time course of the hydrolysis of carboxylic acid p-nitrophenol esters of (R)- and (S)-configurations catalyzed by lipase mutants is monitored spectrophotometrically on microtiter plates, whereby the most enantioselective mutants can be identified quickly. Apart from the fact that no exact ee values are possible, this method is limited to the chiral carboxylic acid class of substances. The same applies to a related test method [L. E. Janes, R. J. Kazlauskas, J. Org. Chem. 1997, 62, 4560-4561]. Also subject to this limitation are related methods which are based on the color change of pH indicators during ester hydrolysis [L. E. Janes, A. C. Lxc3x6wendahl, R. J. Kazlauskas, Chem.xe2x80x94Eur. J. 1998, 4, 2324-2331]. A totally different approach for the identification of chiral catalysts is based on infrared thermography [M. T. Reetz, M. H. Becker, K. M. Kxc3xchling, A. Holzwarth, Angew. Chem. 1998, 110, 2792-2795; Angew. Chem., Int. Ed. 1998, 37, 2547-2650]. However, the further development of this method to enable the quantitative analysis of enantioselective reactions still remains to be done.
The present invention remedies these defects by employing partially or completely isotope-labeled substrates or substrates having an isotope distribution which deviates from the natural distribution for kinetic resolution processes or for stereoselective reactions with prochiral substrates containing enantiotopic groups. This permits the use of an isotope-specific detection system, for example, a mass-spectrometric ionization method, for the quantitative determination of the conversion or the relative proportions of the pseudo-enantiomers or of enantiomeric excess.
As compared to previous approaches, the present invention offers the following advantages:
1) Exact determination of the ee values of kinetic resolution processes and of asymmetrically proceeding conversions of prochiral compounds bearing enantiotopic groups, no limitations being made with respect to the class of substances or the type of reaction.
2) Exact determination of the conversion of the reactions mentioned under 1).
3) Rapid or high throughput testing of the data mentioned under 1) and 2), at least 1000 determinations per day being possible in particular.
The detection systems used in the present invention are mass spectrometers, especially those using electro-spray ionization (ESI) [J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Science (Washington, D.C.) 1989, 246, 64-71] or matrix-assisted laser desorption/ionization (MALDI) [a) K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 1988, 2, 151-153; b) M. Karas, F. Hillenkamp, Anal. Chem. 1988, 60, 2299-2301]. In connection with automated sampler (use of one or more sample charging robots and microtiter plates), optionally with the use of several spectrometers, the method according to the invention is suitable as a high throughput screening method.
The method can be used for finding or optimizing chiral catalysts or chiral agents for asymmetrically proceeding reactions. These include:
a) chiral catalysts or chiral agents for the kinetic resolution of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, and their derivatives;
b) chiral catalysts (e.g., chiral homogeneous or chirally modified heterogeneous catalysts, chiral metal complexes) or chiral agents for the stereoselective conversion of prochiral compounds whose enantiotopic groups include one or more functional groups from the classes of substances of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, or their derivatives;
c) biocatalysts, e.g., enzymes, antibodies, proteins, hormones, phages, ribozymes, peptides or other biopolymers, for the kinetic optical resolution of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, and their derivatives, and for the stereoselective conversion of prochiral compounds whose enantiotopic groups include one or more functional groups from the classes of substances of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, or their derivatives.
The principle underlying the invention is based on the use of isotope-labeled substrates in the form of pseudo-enantiomers or pseudo-prochiral compounds, as represented in Scheme 1.
If one enantiomeric form in a conventional racemate is isotope-labeled, such compounds are called pseudo-enantiomers. If one enantiotopic group of a prochiral substrate is labeled with isotopes, the compound is called pseudo-prochiral, e.g., pseudo-meso. Depending on what types of enantioselective conversions are to be examined or tested according to the invention, different situations are relevant, such as case a, case b, case c and case d in Scheme 1. In kinetic optical resolution processes of any chiral compounds, 1 and 2, which differ in absolute configuration and in the isotope labeling in the functional groups FG*, are prepared in an optically pure form and mixed in a ratio of 1:1 to simulate a racemate (Scheme 1a). After an enantioselective conversion in which the chemical reaction occurs at the functional group (up to a conversion of 50% in the ideal case of a kinetic optical resolution), true enantiomers 3 and 4 are formed along with unlabeled and labeled achiral by-products 5 and 6, respectively.
The ratios of the intensities of 1/2 and 5/6 in the mass spectra (m/z intensities of the quasi-molecular ions) allow for the quantitative determination of the enantioselectivity (ee values) and of the conversion. Optionally, an internal standard may be used according to the invention. According to circumstances, it may be advantageous to effect the isotope-labeling not in the functional group but in the residue R2 of the substrate as outlined in Scheme 1b. In this case, a new pair of pseudo-enantiomers 3/8 is produced (Scheme 1b), the enantioselectivity and conversion being established according to the invention by measuring the m/z intensities of the quasi-molecular ions of 1/7 and 3/8. Thus, the so-called selectivity factors (s or E values) are automatically accessible in both cases [H. B. Kagan, J. C. Fiaud in Top. Stereochem., Vol. 18 (Eds.: E. L. Eliel, S. H. Wilen), Wiley, N.Y., 1988, p. 249-330].
In the case of prochiral substrates having enantiotopic groups, the synthesis of a single pseudo-prochiral compound is required for the screening system according to the invention. When the relevant substrate is a meso-compound, a corresponding meso-compound 9 is first prepared since the stereo-differentiating reaction to be examined yields a mixture of two MS-detectable pseudo-enantiomers 10 and 11 (Scheme 1c). According to the invention, an analogous procedure applies to other prochiral substrates having enantiotopic groups, such as the use of pseudo-prochiral substrates of type 12 (Scheme 1d).
The FG units in Scheme 1 may be a wide variety of functional groups of organic chemistry. Typical representatives are acyloxy residues (xe2x80x94OC(O)R), thioacyloxy residues (xe2x80x94SC(O)R), amido residues (xe2x80x94NHC(O)R), carboxy residues (xe2x80x94CO2R) in the case of cleavage reactions, such as hydrolyses, and further hydroxy residues (xe2x80x94OH), thiol residues (xe2x80x94SH), amino residues (xe2x80x94NH2 or xe2x80x94NHR), or carboxy residues (xe2x80x94CO2H) in the case of bond-forming reactions, such as acylations or esterifications.
Scheme 1 only serves to illustrate or describe the method according to the invention and does not limit it in any way. Rather, any classes of substances and types of reactions are possible as long as the substrates are either chiral, as naturally in a kinetic optical resolution, or they are prochiral and contain enantiotopic groups.
In order to ensure a high sample throughput, the invention provides a design of equipment as outlined in an illustrative way in Scheme 2. With this combination of commercially available devices or parts of apparatus, it is possible to perform at least 1000 ee determinations per day with an accuracy of xc2x15%.